Unlike many foodstuffs such as meat, fish and poultry which have a short shelflife primarily due to their high water content or high water activity and their elevated rate of deterioration except at very low holding temperatures, fruits, vegetables, grains and nuts are often harvested and held for many months prior to processing and conversion into finished food products. However very considerable amounts of stored foodstuffs are lost due to    a) the action of microbial contamination during storage which cause a variety of rots and/or    b) the production of secondary toxic metabolites (aflatoxins) which are harmful to the health of both humans and animals and, once formed are difficult and/or expensive to remove.
The problem with the former is that they are directly caused by microbial contamination of the crop at harvest or at storage and remain undetected until the crop is removed from storage for processing into food materials and products or are resistant to attempts to remove it during the further processing stages of manufacturing. While stored crops are often held in modified atmospheres or forced air flows to make the atmospheric conditions and humidity levels less suited to microbial growth and consequent stored product deterioration, these approaches merely reduce the rate of spread of contamination and have little effect on the total level of contamination certainly from the incoming crop.
Aflatoxin contamination is a result of microbial infection, primarily fungal. Aflatoxins are metabolites of microbial growth and what makes them particularly dangerous is that once generated they remain accumulated in the fruit, nut, grain or vegetable long after any signs of the original infection may have disappeared. They cause a range of carcinogenic disease in both humans and animals including liver cancer, heart failure and renal damage at concentrations as low as 20-30 ug/kg. (The range of aflatoxins causing aflatoxicosis and the systems and mechanisms of action have been extensively studied; the reader is directed to Urbanek (1997) for a more detailed explanation).
Another problem with microbial contamination and subsequent aflatoxin formation is that it is extremely variable. Infections may be very localised so that one fruit or nut may have a very heavy contamination while adjacent fruits may have little or none. A number of attempts have been made to develop rapid methods for identifying contaminated product. The most widely used of these are fluorescence techniques. It has been known for several decades (Shotwell et al, 1972) that on exposure to certain wavelengths of longwave UltraViolet (350 nm to 400 nm) different aflatoxins fluoresce different colours. The so-called B aflatoxins (primarily B1 and B2) fluoresce blue while the so-called G aflatoxins (primarily G1 and G2) fluoresce green. Further metabolised variants of the B aflatoxins, found almost exclusively in milk and milk products, are the so-called M aflatoxins (M1 and M2) fluoresce a mauve blue colour. Although to date some 17 compounds have been designated as aflatoxins, the descriptive term aflatoxin is principally used to describe the 4 furocoumarin metabolites B1, B2, G1, G2 and the two milk variants M1 and M2.
However the fluorescence technique is far from foolproof and false positives and variable performance both between and within different crop groups are a major problem. For example, while Steiner et al, 1988 found that removing all fluorescing figs from a 56 Kg batch reduced the original aflatoxin contamination level from 22.6 to 0.3 ppb, Waked, 1984 found that in cotton seed 92% of fluorescing seeds contained aflatoxin while 8% of non-fluorescing seeds also contained aflatoxin. Similarly, Shotwell et al, 1975 found that after removing all fluorescing kernels and particles from batches of sweetcorn, cracking the corn and then re-inspecting with longwave UV revealed a further 19% of the crop had levels of aflatoxin greater than 3 ppb.
Several patents for detection of aflatoxin in crop specific product have been granted, however they all appear to use the same basic principle of illumination using long wavelength UV combined with manual or automated identification and isolation of fluorescing material. Typical of this is U.S. Pat. No. 4,535,248 used for detecting aflatoxin in almonds. Again while they undoubtedly cause a reduction in total crop aflatoxin levels they do not eliminate it and further expensive remedial action is often necessary before the product is safe to release into the human food chain. Also, these methods are crop specific and practical application is restricted to only a few of them.
Although making a significant improvement to the detection of contaminated harvested crops, the technique is variable in performance and far from reproducible. To this end, manufacturers and further processors have tended to rely on chemical measurement techniques to establish/confirm the cleanness of the manufactured product. Accurate batch sampling of large volumes of incoming material is notoriously difficult to do, is expensive and time consuming and no way guarantees that the product will emerge from the manufacturing process in an aflatoxin free condition. This therefore causes still further expense and delay while the finished product is similarly re-analyzed. Should any contaminated product be discovered at this stage (pathogen or aflatoxin), it necessitates further expensive reprocessing to inactivate the aflatoxin.
Numerous attempts have been made to introduce routine decontamination and/or detoxification techniques as part of the manufacturing process; these may be chemical or physical in nature or a combination of both. However such techniques are either very expensive and only warranted for that part of the crop which could be designed as premium product and thus generate sufficient margin to make its further processing cost effective or they cause the partial degradation of the product quality and thus depress the selling capability or they are only partially effective.
Examples of such techniques include the use of Ozone without Ultraviolet, U.S. Pat. No. 6,294,211, Ozone with short wave UV, Ultrasound, U.S. Pat. No. 5,498,431. U.S. Pat. No. 6,294,211 contains an extensive reference of patents using Ozone either in isolation or in addition with other components to achieve this decontamination effect while U.S. Pat. No. 6,171,625 lists an extensive reference of patented intellectual property utilizing a range of physical and chemical processes used for aflatoxin detoxification including heating, proprionic acid, sodium hydroxide, Fullers earth, aluminosilicate-based clays, ammonia and ammonium compounds. While most are carried out under normal atmospheric conditions, some are under vacuum. Chapman in U.S. Pat. No. 5,082,679 describes and cites numerous techniques for detoxification in an aqueous medium including ammonium compounds and methylamine, and reference is made to all three patents. However most of these treatments can or do have effects on the foodstuff which leads to a reduction in some measure of their quality attributes usually due to either oxidative and/or hydrolytic reactions within or upon the surface of the foodstuff.
An alternative approach has been the use of physical media such as pulsed light in conjunction with photosensitisers such as riboflavin or isoalloxazine (Hlavinka, 2001, Doyle et al 1982).The inactivation of the pathogen occurs by interfering with replication but this does not inactivate the aflatoxin and, as has been shown elsewhere with other pathogens such as E. coli, rupture of the pathogenic organism may lead to an excessive release of toxin and a resultant more acute toxicosis.
Radiant energies have also been used. These may be non-irradiating sources such as Infra-red, visible light and Ultraviolet or irradiating sources such as gamma rays and all have been shown in isolation to be partially capable of reducing overall microbial pathogenicity but with only minimal or nil effect on aflatoxin levels (Van Dyke et al, 1982). Nagarj et al (1985) reduced aflatoxin levels by 16% through sun drying. Muench and Stein (1986) showed that storing cotton seed with high voltage electrical discharge eliminated aflatoxin formation, principally as a consequence of inhibiting the growth of the causative organism. W. German Patent 1 914 095 describes a method of reducing aflatoxin levels in peanuts by exposing them to wet steam followed by a cold water spray in the presence of ultrasound.
Aflatoxins are very heat stable and can withstand boiling (>100° C. for 3 hours). They can be detoxified in alkaline solutions but are relatively stable in neutral and acid pH. However even in neutral solutions the presence of strong oxidisers such as bisulphites, hypochlorites or peroxides will lead to aflatoxin degradation. Unfortunately this usually only occurs in reasonable time (2 hours) at elevated temperatures of 40° C. or greater at additive concentrations of 1% or greater. British Patent 1 117 573 showed that aflatoxin in peanut could be eliminated by exposure to hydrogen peroxide at a pH of 9.5 or greater. Commonly used fumigants showed no detoxification capabilities (Brekke and Stringfellow, 1978)
Another drawback to effective treatment using such additives is that it frequently requires the foodstuff to have either an elevated moisture content or a high water activity for significant detoxification to occur. Rasic et al (1990) reported a significant detoxification in the presence of acids such as lactic and acetic and Magella and Hafez (1982) showed a similar effect with fermenting yoghurt. However Manabe and Matsuura (1972) reported that although B1 and G1 aflatoxins were 50% decomposed during early stages of miso fermentation, B2 and G2 were unaffected.
Altug et al (1990) reported that 45% of B1 aflatoxin in aqueous phase was degraded within 30 minutes when exposed to a low energy source of UV-C but that neither the presence of bisulphite nor peroxide enhanced this degradation. Nkama and Muller (1988) exposed rice to different natural light intensities and moisture contents at 40° C. Both had an effect on the rate of degradation but there was no apparent synergism. Shantha et al (1978) showed that aflatoxin in ground nut oil can be more effectively destroyed by exposure to sunlight than longwave UV or visible light (from a tungsten lamp). Yousef and Marth (1986) demonstrated that milk containing M1 aflatoxin when exposed to UV energy (254 nm) for 20 mins at 25° C. degraded 61% of that toxin. However the addition of 0.05% hydrogen peroxide increased this degradation to 90%. Bencze and Kiermeier (1972) showed that exposing aflatoxin in solid phase to UV-C (254 nm) irradiation induced varying degrees of aflatoxin inactivation with G1 and B1 but not G2 and B2 aflatoxin. In all cases the effect was directly dependent on the amount of available oxygen.
Maeba et al (1988) showed that B1 and G1 were easily degraded in the presence of 1 mg Ozone/1 within 5 mins at 20° C. while B2 and G2 required 40× more Ozone and a minimum of 60 mins exposure for the same level of detoxification.
An alternative approach to a curative treatment to contamination and possible aflatoxin production is a preventative one. A number of authors have identified naturally occurring and potentially protective components in certain foodstuffs (phytoalexins) which can protect and/or inhibit the initial contamination or limit its development. Most of these compounds are somewhat similar in structure to the aflatoxins being in the general group called isocoumarins. Their existence has been shown for example by Jeandet et al (1995) in grapes, Mercier et al (1993) in carrots and Rodov et al (1992) in some citrus fruits. These and others have investigated the effect of UV-C (principally 254 nm but ranging from 220 nm-280 nm) exposure to the level of phytoalexin production. Overall, the response has been negligible in intact fruits and vegetables, mature fruits and nil at low temperatures. More positive results were generally found after wounding or at the sites of infection but again no phytoalexin response could be induced by UV-C irradiation either with or without incidence of wounding at temperatures below 4° C.
Using the broader range of Ultraviolet exposures incorporating wavelengths, wavelength distributions, energy intensities and the associated physical operating conditions as detailed, we have been able to initiate high levels of natural phytoalexins in all of the listed foodstuffs tested. This has been irrespective of age or physical status including the absence of wounding and/or at low temperatures. We have further demonstrated a generalized protective response which has resulted in very low or nil foodstuff losses due to subsequent infection. We have also been able to demonstrate a corresponding resultant increase in maintained quality attributes and extended shelf-life.
This effect has not been demonstrated with any other physical energy, indeed Maghrabi and el-Sayed (1988) and Schmidt et al (1985) have positively demonstrated that irradiation techniques actually reduce the protective response in several crops.
The highest financial returns are most frequently obtained for natural, often organic, products which are in an uncooked state and therefore not exposed to any ‘kill steps’ during harvest, storage or processing to reduce or eliminate pathogens and/or reduce or eliminate aflatoxins. As a consequence, it is often essential that such products are processed in a dry or low moisture environment and not subjected to wet processes or chemical techniques that introduce or utilise compounds not considered natural, i.e. not normally associated with the product in its normal environment. Nor can they be subjected to thermal energy which would change their composition, state or appearance.
Finally, for any process to be considered practical and effective, it must be able to address either or both issues, pathogens and aflatoxins in either a preventative and/or curative manner specific for that product.
Thus there is an increasing need for a preventative method of decontaminating and/or detoxifying large volumes of fruits, vegetables, grains and nuts both before long term storage in an effort to minimize microbially-induced losses due to denaturation and decomposition and a further need for a combined preventative and curative step to eliminate existing and potential further contamination and aflatoxin accumulations in those materials during their further processing following harvest and storage.
This invention addresses both issues and provides a generic and modular approach which can be adapted and optimised for use with a very wide range of fruits, vegetables, grains and nuts.